Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg⁻¹ for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg⁻¹) from shellfish and fish tissues were 114-119% (as amine aldehyde) and 90-115% (as amide aldehyde) with RSDs for both of ≤ 18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL⁻¹ and the LOQ 4 ng mL⁻¹, which corresponds to 10 μg kg⁻¹ in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required.